As we have shown that glucocorticoid receptor binding affinity is decreased at night in subjects with NA, we hypothesized that the reduction in binding affinity is associated with increased expression of glucocorticoid receptor-beta (GR'). GR' is an alternative form of the glucocorticoid receptor formed by alternative splicing that does not bind ligand and therefore does not become transcriptionally active. Ten subjects with NA, defined as those subjects with asthma who experience an overnight fall in lung function of at least 15%, six subjects with NNA, defined as those subjects who experience < 10% overnight fall in lung function and eight non-asthmatic controls underwent bronchoscopy at 4 pm and 4 am in a random order separated by one week. Airway cell expression of GR' was determined by immunohistochemistry using a monoclonal antibody for GR' and the APAAP technique. Subjects with NA exhibited a significant increase in the percentage of airway cells expressing GR' at 4 am as compared to 4 pm. There were no significant differences in the percentage of cells expressing GR' within the NNA and control groups. Percentage of airway cells expressing GR' in subjects with nocturnal asthma (NA), non-nocturnal asthma (NNA) and non-asthmatic controls. To determine if the expression of GR' is associated with decreased steroid responsiveness, nine subjects with NA, seven subjects with NNA and eight non-asthmatic controls underwent blood sampling at 4 pm and 4 am in a random order separated by one week. Peripheral blood mononuclear cells (PBMC) were isolated from blood and stimulated with phytohemaglutinin. Cells were incubated for 72 hours with hydrocortisone (HC) in concentrations ranging from 10-5 to 10-10M. Proliferation response was measured by 3H thymidine incorporation and calculated as a percentage of the positive control. PBMC from NA exhibited less suppression with HC at 4 am as compared to 4 pm (p=0.0004), whereas the NNA and normal control groups exhibited no such circadian variation (p=0.65 and 0.93, respectively). Future studies will determine if IL-2 and IL-4 can induce reduced steroid responsiveness, and if the airway lymphocyte and/or macrophage is the dominant cell involved in this process (specific aim 1a and 1b). The reduction in steroid responsiveness may be secondary to production of GR', or possibly secondary to increased expression of transcription factors nuclear factor kB and activator protein-1, both of which are beginning to be evaluated in the asthmatic airway during the day and at night. In addition, further evaluation of IL-5 mRNA and protein in the airway and its relationship to eosinophil influx will also be determined.